The normal human colonic epithelium undergoes continuous cycle of renewal with a dynamic equilibrium between proliferation, differentiation and apoptosis. Within the base of each crypt (i.e., a deep indentation formed by involutions of the colonic epithelium), yet to be identified stem cells give rise to progenitor cells that divide rapidly four to six times before differentiation. In the mouse intestine, up to 60% of the approximately 250 epithelial cells in a single crypt divide twice daily, yielding up to 260 new cells. Thus, the intestinal epithelium harbors one of the most rapidly dividing cell types in any mammalian organ. Polarized cells migrate along the crypt's basement membrane towards the apical surface of colonic villi, where the cells undergo differentiation as indicated by shifts in cytoskeletal markers such as cytokeratins, cytoplasmic carbonic anhydrase isozyme II, and the brush border enzyme alkaline phosphatase.
Induction of differentiation is incompletely understood, but appears to result from a concerted interplay between growth factors produced by epithelial and stromal cells and signals from basement membrane components. The basement membrane is synthesized by both epithelial and mesenchymal cells, and it contains numerous fenestrations through which processes of myofibroblasts and/or epithelial cells extend. Little is known about the role of fibroblasts in modulating proliferation and differentiation of colonic epithelial cells except that fibroblasts can prolong their survival. Heterologous cross-talk between epithelial and mesenchyrnal compartments involves basement membrane molecules and paracrine factors. The mesenchymal cells apparently produce as yet undefined growth factors for the epithelial cells.
Normal human colonic epithelial cells have been difficult to maintain in vitro. Thus knowledge about intestinal cell regulation has been derived from studies with cell cultures isolated from experimental animals and human colon cancer-derived cell lines. Normal epithelial cells survive only a few days in culture, which has limited studies of proliferation and differentiation. For example, in Whitehead et al's culture (Whitehead R. H. et al, 1999 Gastroenterology, 117:858–865), normal adult colonic crypt cells were embedded into an acellular collagen gel matrix over a feeder layer of bovine aortic endothelial cells, and grown as isolated islands of cells, which increased their survival for up to 16 days.
Normal human cells grown as isolated cultures in monolayer lose many characteristics of those in situ and often resemble the phenotype of cancer cells. For example, colonic cells immortalized with viral oncogenes lose their typical epithelial morphology and neither polarize nor differentiate, limiting the use of their usefulness for biological studies.
In contrast, cells in a tissue-like context maintain a similar phenotype as those growing in situ. Organotypic reconstructs and cultures can serve as replacement organs, as models for the study of the basic biology of organs, and as screening systems for development of drugs, to identify drug candidates as well as to observe candidate drug activity, such as its transport into organs, or dosage requirements. Obviously, the most useful organotypic cell reconstructs and cultures have a long shelf life and allow the component cells to maintain their normal cellular activities and morphologies and the ability to function within the organ.
Organotypic culture models for esophagus, bladder, pancreatic duct, breast, lung, liver, and human skin have all been used for studies of tissue physiology, drug delivery and transformation. The cells in those organotypic cultures retain many of the functions they had in situ. For example, normal human melanocytes in the epidermis of an organotypic skin culture were shown to develop close adhesive and gap junctional communications with basal layer keratinocytes.
In contrast to cells of most other organs, normal human colon cells have been difficult to maintain in vitro. Currently available normal human intestinal epithelial cells are derived from the small intestine and exhibit undifferentiated features, while differentiated enterocytes remain in culture for only 10–12 days. Models of human intestine in culture are not suitable for studies of proliferation and differentiation. The cultured cells survive for only a few days. Co-culture of intestinal epithelial cells with fibroblasts or myofibroblasts could prolong survival. To improve survival, immortalization of colonic cells with genes from oncogenic viruses has been attempted. However, the transformed cells lost their typical epithelial morphology and did not polarize or differentiate.
There remains a need in the art for compositions and methods that provide a useful source of normal human intestinal epithelial cells which maintains in situ-like properties for use in studies of colon biology, screening for drug absorption and efficacy and for therapeutic uses, such as in transplantation or the treatment of colon lesions.